Discovery of a Novel Potent and Selective HSD17B13 Inhibitor, BI-3231, a Well-Characterized Chemical Probe Available for Open Science

Genome-wide association studies in patients revealed HSD17B13 as a potential new target for the treatment of nonalcoholic steatohepatitis (NASH) and other liver diseases. However, the physiological function and the disease-relevant substrate of HSD17B13 remain unknown. In addition, no suitable chemical probe for HSD17B13 has been published yet. Herein, we report the identification of the novel potent and selective HSD17B13 inhibitor BI-3231. Through high-throughput screening (HTS), using estradiol as substrate, compound 1 was identified and selected for subsequent optimization resulting in compound 45 (BI-3231). In addition to the characterization of compound 45 for its functional, physicochemical, and drug metabolism and pharmacokinetic (DMPK) properties, NAD+ dependency was investigated. To support Open Science, the chemical HSD17B13 probe BI-3231 will be available to the scientific community for free via the opnMe platform, and thus can help to elucidate the pharmacology of HSD17B13.

H NMR spectra for key compounds S61

High-throughput Screening Results
Figure S1. Results from the full-diversity screening campaign for novel HSD17B13 inhibitors.
(A) Histogram for the single-dose experiments of the primary screening campaign. In total, 1.09 million compounds were tested, and a hit rate of 2.1% (total: 22,401 compounds) was observed when applying a hit threshold of 70% residual enzyme activity being indicated by the dashed grey line. (B) Assay quality parameter Z' was monitored throughout the entire screening campaign. Each of the 3.295 compound plates (384-well plate containing 16 high and low controls each) stayed within the predefined quality threshold of Z′ ≥ 0.5. (C) Confirmation of primary screening hits. The average PoC values of duplicate single-dose determinations in the hit confirmation experiments are plotted against PoC values from the primary screen. Compounds are considered as confirmed hits when they lead to an average residual enzyme activity below 75% (= hit cut-offPS + ~1xSDPS). Overall, a confirmation rate of 80% (total: 17,082 compounds) was obtained. Two-fold deviation between runs is indicated by the blue dashed line and the solid line represents the line of identity. SD: standard deviation; PS: primary screen (D) Determination of compound potencies of selected hits from the screening campaign. The average IC50 values from two individual measurements are plotted in decreasing order.

A B C D
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General Methods and Materials
All commercially available chemicals were used as received from their commercial supplier.
Anhydrous solvents were either purchased or prepared according to standard procedures S1 and stored over molecular sieves under argon. Unless stated otherwise, all reactions were carried out in oven-dried (at 120 °C) glassware under an inert atmosphere of argon. A Biotage Initiator Classic microwave reactor was used for reactions conducted in a microwave oven. Reactions were monitored by TLC on aluminum-backed plates coated with Merck Kieselgel 60 F 254 with visualization under UV light at 254 nm, and with HPLC-MS analysis (for HPLC-MS methods, see Table S1). Unless stated otherwise, crude products were purified by flash column chromatography on silica (using a Biotage IsoleraOne, Biotage IsoleraFour or CombiFlash® Teledyne Isco system) or by (semi)-preparative reversed-phase HPLC (Agilent Acquity or Waters instruments). Unless specified otherwise, the purity of all final compounds was determined to be ≥ 95% by LC-MS.
Nuclear magnetic resonance (NMR) spectra were recorded at room temperature ((22 ± 1 °C), on a Bruker Avance 400 spectrometer with tetramethylsilane as an internal reference. Chemical shifts δ are reported in parts per million (ppm). 1 H NMR spectra were referenced to the residual partially non-deuterated solvent signal of DMSO (δ = 2.50 ppm). Coupling constants J are reported in Hz, and splitting patterns are described as br = broad, s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet and m = multiplet. High-resolution mass spectra were recorded on a Thermo Scientific LTQ Orbitrap XL using electrospray ionization in positive ion mode (ESI+).
MarvinSketch software version 20. 19.1 was used to generate compound names. S5

Mobile phase preparations
Examples: -The mobile phase "Water 0.1% TFA (v/v)" is prepared by adding 1 ml of a commercially available TFA solution to 999 ml water.
-The mobile phase "Water 0.1% NH3" is prepared by adding 4 ml of a commercially available concentrated ammonium hydroxide solution (25 wt%) to 996 ml water.

Syntax for column description
Description_Dimensions ID x length_Particle Size Convention: Sections separated by underscores; blanks between numbers and unit; ID and length in mm, particle size in µm; ID and particle size always with one digit e.g.: XBridge C18_4.6 x 50 mm_3.5 µm S8

Synthesis of Compounds 2-11
For a general synthetic strategy for compounds 1-11, see Scheme 1 and Experimental Section in main text.